Monoclonal antibodies (mAbs) specific for free protein S and devoid of reactivity with protein S-C4b-BP complexes, have been produced. A one-step sandwich-type enzyme-linked immunoassay (ELISA) has been developed with two mAbs reacting with distinct epitopes of free protein S. F(ab')2 fragments from mAb 15C4 were coated on microplates and mAb 34G2 conjugated with horseradish peroxidase (HRP) was added immediately before diluted plasma. The presence of calcium in the sample diluent prevented dissociation of complexes during the assay. This assay was specific as demonstrated by good recovery of purified protein S added to plasma and the lack of influence of C4B-BP-protein S complexes. Thus, addition of increasing amounts of purified C4B-BP to human citrated plasma induced a dose-dependent decrease of free protein S. The assay was sensitive, allowing measurement of 5-500 ng/ml of free protein S, with a detection threshold of 2 ng/ml. It was also reproducible with inter-assay and intra-assay variation coefficients of 2.5-5.1% and 3.1-5.0%, respectively. Thus, this new ELISA of free protein S antigen in plasma has the advantages of being fast, accurate and reproducible. It appears to be extremely useful for routine studies as no preliminary treatment of plasma is required.